The general area I research is the intracellular signaling that occurs in response to cell surface receptor activation. In particular, I study inositol 1,4,5-trisphosphate (IP3) receptors, proteins that form ion channels in endoplasmic reticulum (ER) membranes and which, upon the binding of IP3 and Ca2+, mobilize Ca2+ stored in the ER. My long-term objectives are to understand the role of IP3 receptors in intracellular signaling and the mechanisms by which they are regulated, and the mechanism by which signaling proteins are degraded by the ubiquitin / proteasome pathway (UPP). In recent years I have focused on a phenomenon termed "IP3 receptor down-regulation". This is an adaptation to cell surface receptor stimulation that rapidly reduces cellular IP3 receptor content and, thus, the sensitivity of ER Ca2+ stores to IP3. It results from an acceleration of IP3 receptor degradation via the UPP, the route for destruction of many key proteins. Currently, however, very little is known about the molecular details of IP3 receptor down-regulation. The current proposal is designed to rectify this through three Specific Aims. (1) Identification of the ubiquitin-protein ligase that mediates IP3 receptor ubiquitination. The approach taken will be to define the roles of candidate ligases in ubiquitination by inhibiting their expression using RNA interference (RNAi). (2) Identification of the proteins responsible for coupling ubiquitinated IP3 receptors to the proteasome. The approach taken will be to determine which proteins associate with IP3 receptors as they becomes ubiquitinated and then define their roles in coupling IP3 receptors to the proteasome by inhibiting their expression using RNAi. (3) Definition of the events that trigger UN receptor ubiquitination. The approach taken will be to mutate IP3 receptors and manipulate the levels or activities of intracellular signals to identify factors and regions of IP3 receptors that trigger ubiquitination, and then to examine possible structural changes in these regions. Accomplishment of these Aims will both further our understanding of IP3 receptor down-regulation, and provide a paradigm of how ER proteins are degraded by the UPP in mammalian cells. The health relevance of this work is three fold. First, it will lead to a better understanding of the mechanisms that cells use to adapt to extracellular stimuli; such adaptation is the basis for many physiological modifications to cell function and of tolerance to the effects of therapeutic and recreational drugs. Second, IP3 receptor down-regulation occurs in several pathophysiological conditions and, thus, obtaining a deeper understanding of its mechanism is of potential benefit to those suffering from these conditions. Third, because of its pivotal role in controlling cell function, the UPP is being explored as a therapeutic target, particularly in cancer. Obtaining a better understanding of the enzymes and mechanisms used in the UPP will both facilitate drug design and give us a better appreciation of their effects.